Merged Tacrine-Based, Multitarget-Directed Acetylcholinesterase Inhibitors 2015–Present: Synthesis and Biological Activity

Acetylcholinesterase is an important biochemical enzyme in that it controls acetylcholine-mediated neuronal transmission in the central nervous system, contains a unique structure with two binding sites connected by a gorge region, and it has historically been the main pharmacological target for treatment of Alzheimer’s disease. Given the large projected increase in Alzheimer’s disease cases in the coming decades and its complex, multifactorial nature, new drugs that target multiple aspects of the disease at once are needed. Tacrine, the first acetylcholinesterase inhibitor used clinically but withdrawn due to hepatotoxicity concerns, remains an important starting point in research for the development of multitarget-directed acetylcholinesterase inhibitors. This review highlights tacrine-based, multitarget-directed acetylcholinesterase inhibitors published in the literature since 2015 with a specific focus on merged compounds (i.e., compounds where tacrine and a second pharmacophore show significant overlap in structure). The synthesis of these compounds from readily available starting materials is discussed, along with acetylcholinesterase inhibition data, relative to tacrine, and structure activity relationships. Where applicable, molecular modeling, to elucidate key enzyme-inhibitor interactions, and secondary biological activity is highlighted. Of the numerous compounds identified, there is a subset with promising preliminary screening results, which should inspire further development and future research in this field.


Introduction
Acetylcholinesterase (AChE; EC 3.1.1.7) catalyzes the hydrolysis reaction of acetylcholine (ACh) to choline and acetate ( Figure 1A), making it responsible for terminating ACh-mediated synaptic transmission in the central nervous system (CNS) with high catalytic efficiency [1,2]. Structurally, three key features are known: The catalytic active site (CAS), the gorge, and the peripheral anionic site (PAS) ( Figure 1B,C). The CAS is in the interior of the enzyme and is where the hydrolysis reaction takes place. It can be further divided into regions including the esteratic site, which contains the catalytic triad (Ser203, His447, Glu334 in hAChE (PDB:4EY4)), the anionic site, which contains aromatic residues (Trp86, Tyr133, Tyr337, Phe338) that stabilize the quaternary ammonium group of ACh through cation-π interactions, the oxyanion hole, which contains residues (Gly121, Gly122, Ala204) to stabilize the negatively charged transition state, and the acyl pocket, which uses residues (Phe295 and Phe297) to control substrate specificity. The gorge region, having dimensions of about 20 Å long and 5 Å wide, connects the CAS to the PAS and the enzyme exterior. It is lined with primarily aromatic amino acids. The PAS on the exterior of the enzyme also consists of primarily aromatic residues (Tyr72,  [3], with permission from Elsevier). (C) Surface representation of the hAChE (PDB:4EY4) active site looking down the gorge with regions color coded as in (B) (reprinted from [3], with permission from Elsevier).
AChE has received considerable attention as a target of chemical intervention, particularly for therapeutic treatment of Alzheimer's disease (AD). In the United States, AD is the sixth leading cause of death and affects nearly 6 million people, and that number is estimated to grow to nearly 14 million in the next few decades. The disease is characterized by progressive neurodegeneration and diverse symptoms, the most noticeable being cognitive failure (memory loss, language difficulties, inability to plan or problem solve) [10]. The connection between AChE and AD dates back approximately 40 years and is based on the role of ACh in learning and memory and the observation of reduced cholinergic function in the brains of AD patients [11][12][13][14][15][16]. The logical progression has been to develop an AChE inhibitor(s) (AChEi) to slow the breakdown of ACh and boost its levels (cholinergic hypothesis). Indeed, over the last several decades, four AChEi (tacrine, donepezil, rivastigmine, and galantamine) have all been used to treat AD ( Figure 2). A fifth drug used to treat AD, memantine, functions as an N-methyl-D-aspartate receptor (NMDAR) antagonist, and it is often used in combination with donepezil. While these drugs have had moderate success in alleviating cognitive symptoms, they are incapable of halting progression or curing AD [17]. This is thought to be, in large part, due to the complex nature and etiology of AD, and it is largely accepted that new and improved AD drugs should target additional factors beyond ACh.   [3], with permission from Elsevier). (C) Surface representation of the hAChE (PDB:4EY4) active site looking down the gorge with regions color coded as in (B) (reprinted from [3], with permission from Elsevier).
AChE has received considerable attention as a target of chemical intervention, particularly for therapeutic treatment of Alzheimer's disease (AD). In the United States, AD is the sixth leading cause of death and affects nearly 6 million people, and that number is estimated to grow to nearly 14 million in the next few decades. The disease is characterized by progressive neurodegeneration and diverse symptoms, the most noticeable being cognitive failure (memory loss, language difficulties, inability to plan or problem solve) [10]. The connection between AChE and AD dates back approximately 40 years and is based on the role of ACh in learning and memory and the observation of reduced cholinergic function in the brains of AD patients [11][12][13][14][15][16]. The logical progression has been to develop an AChE inhibitor(s) (AChEi) to slow the breakdown of ACh and boost its levels (cholinergic hypothesis). Indeed, over the last several decades, four AChEi (tacrine, donepezil, rivastigmine, and galantamine) have all been used to treat AD ( Figure 2). A fifth drug used to treat AD, memantine, functions as an N-methyl-D-aspartate receptor (NMDAR) antagonist, and it is often used in combination with donepezil. While these drugs have had moderate success in alleviating cognitive symptoms, they are incapable of halting progression or curing AD [17]. This is thought to be, in large part, due to the complex nature and etiology of AD, and it is largely accepted that new and improved AD drugs should target additional factors beyond ACh.
As mentioned above, various other factors are thought to play a role in the AD onset and progression, and there is a significant amount of interplay among these factors and with AChE. The amyloid hypothesis focuses on extracellular aggregates of the amyloid-β (Aβ) peptide in the form of oligomers and plaques, which disrupt synaptic transmission and cause neuronal death. The Aβ peptide itself is generated by β-secretase (BACE1) and γ-secretase cleavage of the amyloid precursor protein (APP) [18][19][20][21][22]. Aβ aggregation is promoted through an interaction with the PAS of AChE [23][24][25] and with metal ions (Cu 2+ and Zn 2+ ) [26][27][28]. These metal ions may also, alone or in combination with Aβ oligomers, lead to the production of reactive oxygen species (ROS) and inflammation, which have been implicated in neuronal death and AD progression [29][30][31]. Dyshomeostasis of other intracellular metal ions, such as Ca 2+ , may also contribute to neurodegeneration [32,33]. Additionally, metabolic enzymes such as 15-lipoxygenase , which catalyzes the oxidation of polyunsaturated fatty acids to hydroperoxy acids, and cyclooxygenase-2 (COX-2), which is involved in the conversion of arachidonic acid to prostaglandins, contribute to ROS, oxidative stress, and inflammation [34][35][36][37]. Monoamine oxidase (MAO) is also of interest as the increase of monoaminergic neurotransmission through inhibition may alleviate AD symptoms, and ROS-byproducts of the MAO reaction exacerbate oxidative stress [38,39]. Carbonic anhydrase (CA) has even been implicated in AD, and modulation may be beneficial for AD [40,41]. Furthermore, the tau hypothesis focuses on abnormal phosphorylation of the tau protein. This protein is associated with microtubules and interacts with the neuronal cytoskeleton to facilitate intracellular signaling. When hyperphosphorylated, tau can aggregate to form intracellular neurofibrillary tangles (NFTs). This leads to compromised axonal transport and diminished synaptic function, and there is evidence that tau may act synergistically with Aβ [18,42]. Finally, the mitochondrial cascade hypothesis of AD suggests that the individual genetic makeup determines a baseline mitochondrial function, and the genetics plus environment determines how the mitochondrial function declines with age. The declining mitochondrial function can then initiate other pathologies associated with AD, such as Aβ aggregation and tau phosphorylation [43,44]. active site with key amino acid residues color coded by region (reprinted from [3], with permission from Elsevier). (C) Surface representation of the hAChE (PDB:4EY4) active site looking down the gorge with regions color coded as in (B) (reprinted from [3], with permission from Elsevier).
AChE has received considerable attention as a target of chemical intervention, particularly for therapeutic treatment of Alzheimer's disease (AD). In the United States, AD is the sixth leading cause of death and affects nearly 6 million people, and that number is estimated to grow to nearly 14 million in the next few decades. The disease is characterized by progressive neurodegeneration and diverse symptoms, the most noticeable being cognitive failure (memory loss, language difficulties, inability to plan or problem solve) [10]. The connection between AChE and AD dates back approximately 40 years and is based on the role of ACh in learning and memory and the observation of reduced cholinergic function in the brains of AD patients [11][12][13][14][15][16]. The logical progression has been to develop an AChE inhibitor(s) (AChEi) to slow the breakdown of ACh and boost its levels (cholinergic hypothesis). Indeed, over the last several decades, four AChEi (tacrine, donepezil, rivastigmine, and galantamine) have all been used to treat AD ( Figure 2). A fifth drug used to treat AD, memantine, functions as an N-methyl-D-aspartate receptor (NMDAR) antagonist, and it is often used in combination with donepezil. While these drugs have had moderate success in alleviating cognitive symptoms, they are incapable of halting progression or curing AD [17]. This is thought to be, in large part, due to the complex nature and etiology of AD, and it is largely accepted that new and improved AD drugs should target additional factors beyond ACh.  Tacrine was the first AChEi approved by the United States Food and Drug Administration for the treatment of AD in 1993. However, it is no longer used clinically due to poor pharmacokinetics requiring four times per day dosing and side effects, the most notorious being hepatotoxicity. Structurally, it is a tricyclic molecule (see Figure 2) consisting of an aromatic A-ring, heteroaromatic B-ring containing an amino substituent at position 9, and a saturated C-ring. Hepatotoxicity has been linked to A-ring hydroxylation during metabolism, which is followed by subsequent conversion to a reactive quinone-like metabolite [45][46][47][48]. Tacrine has, however, remained an important molecule for research into new AD drugs. This is largely due to its strong inhibition of AChE (low nanomolar IC 50 ), ligand efficiency, synthetic accessibility, tolerance of structural modification, and suitability as a starting point for the multitarget-directed ligand (MTDL) strategy. As mentioned above, AD is multifactorial in nature and new and improved AD drugs should target additional factors beyond the cholinergic system. The MTDL strategy for AD, which aims to combine two or more pharmacophores into a single chemical entity capable of acting on multiple aspects of AD at once, has been implemented using tacrine with varying degrees of success for nearly three decades [49][50][51][52][53][54]. It should be noted that the MTDL strategy is not unique to AD. For example, it has been applied to the treatment of cancer, malaria, and diabetes [55,56].
Morphy and Rankovic [57] classified MTDLs based on the degree of overlap of the pharmacophores ( Figure 3). "Linked" MTDLs contain well separated pharmacophores joined by a distinct linker region that is not present in either parent compound. "Merged" MTDLs represent the opposite end of a continuum where there is a high degree of overlap between the pharmacophores based on structural similarities in the parent compounds. The merged approach often results in smaller and simpler molecules, whereas the linked approach leads to larger, higher molecular weight molecules. The tacrine-based MTDLs reported in the literature are numerous, and our initial survey led us to divide tacrine-based MTDLs into two groups: Linked and merged. To keep the scope of this manuscript manageable, we report herein only on the merged MTDLs. Furthermore, to focus on current advances, this review details only compounds that were newly reported in the last five years (2015-present). While we recognize the significance of linked tacrine-based MTDLs to this field, we feel that a detailed discussion warrants a separate report, which will be composed in due course. (2015-present). While we recognize the significance of linked tacrine-based MTDLs to this field, we feel that a detailed discussion warrants a separate report, which will be composed in due course.

Figure 3.
Tacrine-based multitarget-directed ligand (MTDLs) can be classified as "linked" or "merged" based on the degree of overlap of the pharmacophores (adapted with permission from [57], copyright (2005) American Chemical Society).
The merged tacrine-based MTDLs discussed have been grouped based on structural similarities. Special emphasis is placed on the synthesis of the compounds and comparison of in vitro AChE inhibition to tacrine, 6-chlorotacrine, or 7-methoxytacrine as appropriate. The 6-chloro substituent on the tacrine scaffold is known to improve the inhibition of AChE while increasing toxicity, and the 7methoxy substituent, conversely, is known to weaken inhibition while reducing toxicity [58,59]. Unless otherwise stated, inhibition is evaluated using the classic Ellman method [60] with AChE from either Electrophorus electricus (EeAChE) or human (hAChE). These two enzymes have a 56.6% sequence identity, but EeAChE is generally the less expensive choice for preliminary testing [61]. We highlight key structure-activity relationship (SAR) trends and in silico molecular modeling interactions where appropriate, while also describing relevant secondary in vitro and in vivo biological activity. Molecular modeling uses either hAChE or Torpedo californica AChE (TcAChE), which have a 51.9% sequence identity [61]. Together, the in vitro, in silico, and in vivo data represent an "in combo" approach to AD drug discovery [56,62].

Pyranopyrazole Tacrines
One of the most studied modifications to the tacrine core over the last five years has been the replacement of the aromatic A-ring with a fused pyranopyrazole moiety. The resulting 76 different analogs of this tetracyclic scaffold can be divided among two series: 1a-z, varying in the 4-aryl substituent on the pyran ring, and 2a-ax, varying at four positions around the scaffold (Scheme 1A) [63][64][65][66][67]. Synthetically, pyranopyrazole tacrines are accessed via closely related two-step sequences (Scheme 1B,C). The pyrano [2,3-c]pyrazole core can be constructed using a one-pot four-component reaction between β-ketoesters, hydrazine hydrate or hydrazine derivatives, malononitrile, and aryl or alkyl aldehydes under ultrasonic irradiation in the presence of (S)-Pro to give 3a-n or 4a-o, t-aa, ae, af [63,64,66]. Alternatively, the pyrano[2,3-c]pyrazole core 3o-z or 4ag-ax can be constructed from the reaction of 3-methyl-1H-pyrazol-5(4H)-one or 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one with aryl aldehydes and malononitrile [65,67]. A subsequent Friedländer reaction with the appropriate cyclohexanone, cycloheptanone, or tetrahydro-4H-thiopyran-4-one and AlCl3 gave the target compounds 1a-z and 2a-ax as racemic mixtures. The merged tacrine-based MTDLs discussed have been grouped based on structural similarities. Special emphasis is placed on the synthesis of the compounds and comparison of in vitro AChE inhibition to tacrine, 6-chlorotacrine, or 7-methoxytacrine as appropriate. The 6-chloro substituent on the tacrine scaffold is known to improve the inhibition of AChE while increasing toxicity, and the 7-methoxy substituent, conversely, is known to weaken inhibition while reducing toxicity [58,59]. Unless otherwise stated, inhibition is evaluated using the classic Ellman method [60] with AChE from either Electrophorus electricus (EeAChE) or human (hAChE). These two enzymes have a 56.6% sequence identity, but EeAChE is generally the less expensive choice for preliminary testing [61]. We highlight key structure-activity relationship (SAR) trends and in silico molecular modeling interactions where appropriate, while also describing relevant secondary in vitro and in vivo biological activity. Molecular modeling uses either hAChE or Torpedo californica AChE (TcAChE), which have a 51.9% sequence identity [61]. Together, the in vitro, in silico, and in vivo data represent an "in combo" approach to AD drug discovery [56,62].
In addition to acting as AChEi, pyranopyrazole tacrines have shown activity against ROS and Aβ. Among compounds reported by Khoobi et al., the most active inhibitor 1h (EeAChE IC 50 = 190 nM) showed a slight improvement over tacrine (EeAChE IC 50 = 280 nM) ( Table 1). SAR showed that a methoxy substituent at the 4-position of the phenyl ring showed better inhibition than methyl or fluoro substituents, and a second methoxy group at the 3-position, as seen in 1h, further improved potency. The Lineweaver-Burk plot showed varying x-and y-intercepts indicating a mixed-type inhibition for this compound (i.e., binding to both the substrate active site (CAS) and a second distinct site (PAS)). Molecular modeling predicted the R-enantiomer of 1h to interact with TcAChE near the CAS. The methoxyphenyl group showed a key hydrogen bonding interaction with His440 of the catalytic triad. However, the S-enantiomer was predicted to interact with the top of the gorge near the PAS. At 10 µM, 1h showed similar neuroprotection to quercetin in response to H 2 O 2 -induced damage in PC12 cells [63]. Chioua et al. identified two promising EeAChEi in the 4-nitrophenyl analog 1r (IC 50 = 170 nM), which was comparable to tacrine (IC 50 = 190 nM), and the 2-methoxyphenyl analog 1f (IC 50 = 1.52 µM), which was 8-fold less potent than tacrine (Table 1). Interestingly, the Lineweaver-Burk plot indicated that 1f was a noncompetitive inhibitor, while molecular modeling of (R)-and (S)-1r with EeAChE showed similar interactions confined to the PAS, namely π-π stacking interactions between the pyranopyrazole moiety and Trp286 and the pyridine moiety and Tyr341. Furthermore, both 1f and 1r showed complete inhibition of Aβ 1-40 EeAChE-induced aggregation at 25 µM, and they showed comparable neuroprotection against oligomycin A/rotenone-induced oxidative stress in cortical neurons. However, only 1f was nontoxic to HepG2 cells at 300 µM and showed favorable in silico CNS permeability [64]. Compounds 1s, with a para-thioanisole substituent, and 1x, with a biphenyl substituent, were also highly potent EeAChEi being 4.5-to 6-fold more potent than tacrine and having IC 50 = 58 and 44 nM, respectively (Table 1). Molecular modeling indicated that both enantiomers of 1x interacted with TcAChE at the gorge entrance with the biphenyl substituent positioned towards the CAS [65]. Additional studies have focused on varying ring size on the tacrine core and side chains off of the pyrano[2,3-c]pyrazole moiety [66,67]. Compounds 2a-af, with one exception, all showed IC 50 < 2 µM (EeAChE). SAR results showed that a propyl or phenyl substituent at R 1 and a 4-methoxyphenyl or 4-fluorophenyl substituent at R 3 gave the best inhibition. Expansion of the tacrine cyclohexane to a cycloheptane gave mixed inhibition results, and bioisosteric replacement of a cyclic methylene with S had little effect. Of note, compared to tacrine (IC 50 = 221 nM), 2u (IC 50 = 34 nM) and 2x (IC 50 = 81 nM) were 6-and 3-fold more potent, respectively. Molecular modeling of 2u with TcAChE ( Figure 4) showed the pyrazole ring forming key H-bonds with Glu199 and Tyr130 and π-π stacking with Trp84 in the CAS. Additionally, the 4-fluorophenyl moiety was positioned towards the oxyanion hole forming an amide-π stacking interaction with Gly119. The Lineweaver-Burk plot showed a mixed-type inhibition for 2u. Moreover, 2x was a moderate inhibitor of 15-LOX (IC 50 = 31 µM, compared to quercetin IC 50 = 18 µM), while 2u showed no inhibition of this enzyme, and both 2u and 2x were less hepatotoxic than tacrine, showing no appreciable change in the HepG2 cell viability up to 50 µM [66]. Compounds 2ag-ax were overall less potent inhibitors of AChE. Of this series, 2ag and 2ah with a phenyl and 4-methoxyphenyl substituent off the pyran, respectively, showed the best inhibition of EeAChE with IC 50~2 .8 µM [67]. In addition to acting as AChEi, pyranopyrazole tacrines have shown activity against ROS and Aβ. Among compounds reported by Khoobi et al., the most active inhibitor 1h (EeAChE IC50 = 190 nM) showed a slight improvement over tacrine (EeAChE IC50 = 280 nM) ( Table 1). SAR showed that a methoxy substituent at the 4-position of the phenyl ring showed better inhibition than methyl or fluoro substituents, and a second methoxy group at the 3-position, as seen in 1h, further improved potency. The Lineweaver-Burk plot showed varying x-and y-intercepts indicating a mixed-type inhibition for this compound (i.e., binding to both the substrate active site (CAS) and a second distinct site (PAS)). Molecular modeling predicted the R-enantiomer of 1h to interact with TcAChE near the CAS. The methoxyphenyl group showed a key hydrogen bonding interaction with His440 of the   [66], with permission from Elsevier). The pyrazole ring forms key H-bonds with Glu199 and Tyr130 and π-π stacking with Trp84. The 4-fluorophenyl moiety forms an amide-π stacking interaction with Gly119.

Pyranopyranone Tacrines
There have also been recent reports of pyranopyranone tacrines 5a-o and 6a-l (Scheme 2A) [68,69]. These compounds contain hydroxypyranone moieties that offer activity against ROS, while maintaining a potent inhibition of AChE. The target compounds were prepared in two steps from a starting hydroxypyranone (Scheme 2B,C). 4-Hydroxy-6-methyl-2H-pyran-2-one or kojic acid (KA), a natural fungal metabolite with ROS scavenging ability [70], were reacted with aryl aldehydes and malononitrile in the presence of DABCO or Et3N to afford intermediate aminocarbonitriles 7a-o and 8a-l. Then, the Friedländer reaction with cyclohexanone in the presence of AlCl3 gave the target compounds 5a-o and 6a-l as racemic mixtures [68,69].   [66], with permission from Elsevier). The pyrazole ring forms key H-bonds with Glu199 and Tyr130 and π-π stacking with Trp84. The 4-fluorophenyl moiety forms an amide-π stacking interaction with Gly119.

Pyranopyranone Tacrines
There have also been recent reports of pyranopyranone tacrines 5a-o and 6a-l (Scheme 2A) [68,69]. These compounds contain hydroxypyranone moieties that offer activity against ROS, while maintaining a potent inhibition of AChE. The target compounds were prepared in two steps from a starting hydroxypyranone (Scheme 2B,C). 4-Hydroxy-6-methyl-2H-pyran-2-one or kojic acid (KA), a natural fungal metabolite with ROS scavenging ability [70], were reacted with aryl aldehydes and malononitrile in the presence of DABCO or Et 3 N to afford intermediate aminocarbonitriles 7a-o and 8a-l. Then, the Friedländer reaction with cyclohexanone in the presence of AlCl 3 gave the target compounds 5a-o and 6a-l as racemic mixtures [68,69].   [66], with permission from Elsevier). The pyrazole ring forms key H-bonds with Glu199 and Tyr130 and π-π stacking with Trp84. The 4-fluorophenyl moiety forms an amide-π stacking interaction with Gly119.

Pyranopyranone Tacrines
There have also been recent reports of pyranopyranone tacrines 5a-o and 6a-l (Scheme 2A) [68,69]. These compounds contain hydroxypyranone moieties that offer activity against ROS, while maintaining a potent inhibition of AChE. The target compounds were prepared in two steps from a starting hydroxypyranone (Scheme 2B,C). 4-Hydroxy-6-methyl-2H-pyran-2-one or kojic acid (KA), a natural fungal metabolite with ROS scavenging ability [70], were reacted with aryl aldehydes and malononitrile in the presence of DABCO or Et3N to afford intermediate aminocarbonitriles 7a-o and 8a-l. Then, the Friedländer reaction with cyclohexanone in the presence of AlCl3 gave the target compounds 5a-o and 6a-l as racemic mixtures [68,69]. Compared to tacrine (EeAChE IC 50 = 48 nM), 5a-o showed a weaker inhibition. The most potent compound 5f with a 2,3-dichlorophenyl group was 8-fold weaker (EeAChE IC 50 = 370 nm). 5f also showed moderate neuroprotection in PC12 cells against H 2 O 2 -induced damage, but it did not perform as well as quercetin in the same assay. The Lineweaver-Burk plot of 5f showed a mixed inhibition, suggesting an interaction with both the CAS and PAS. Interestingly, molecular modeling showed that the R-enantiomer of 5f was predicted to interact with the PAS, while the S-enantiomer was predicted to interact with the mid-gorge region and span the PAS-CAS distance [68]. Similarly, 6a-l also showed a comparatively weaker inhibition. Of this series, the most potent inhibitor of EeAChE was the 3-methoxyphenyl substituted 6d (IC 50 = 640 nM), which was 20-fold less potent than tacrine (IC 50 = 31 nM). Promisingly though, most of these compounds were significantly less toxic than tacrine. In addition, 6d maintained a 65.5% viability in HepG2 cells at 1 mM, 6-fold less toxic than tacrine (10.9% viability at 1 mM). Moreover, 6d improved upon the known antioxidant capacity of KA (ORAC assay; 4.79 and 2.51 Trolox equivalents for 6d and KA, respectively), and it showed a capable neuroprotection against oligomycin/rotenone and Aβ 1-40 in SH-SY5Ycells at 3 µM. Molecular modeling of hAChE with 6d Rand S-enantiomers indicated an association of both with the PAS via π-π stacking with Trp286 and Tyr341, acting as a barrier to the active site of the enzyme ( Figure 5). Additional key H-bonds were noted, including the primary hydroxyl group for both enantiomers with Tyr72 and the tacrine-like amino group with Tyr124 (R-enantiomer) or Asp74 (S-enantiomer) [69]. conditions: (i) DABCO (rt, 12 h, quant.) or Et3N (Δ, 5 min, 63-77%), EtOH, rt; (ii) cyclohexanone, AlCl3, DCE or 1,4-dioxane, Δ, 2-18 h, 50-87%.
Compared to tacrine (EeAChE IC50 = 48 nM), 5a-o showed a weaker inhibition. The most potent compound 5f with a 2,3-dichlorophenyl group was 8-fold weaker (EeAChE IC50 = 370 nm). 5f also showed moderate neuroprotection in PC12 cells against H2O2-induced damage, but it did not perform as well as quercetin in the same assay. The Lineweaver-Burk plot of 5f showed a mixed inhibition, suggesting an interaction with both the CAS and PAS. Interestingly, molecular modeling showed that the R-enantiomer of 5f was predicted to interact with the PAS, while the S-enantiomer was predicted to interact with the mid-gorge region and span the PAS-CAS distance [68]. Similarly, 6a-l also showed a comparatively weaker inhibition. Of this series, the most potent inhibitor of EeAChE was the 3-methoxyphenyl substituted 6d (IC50 = 640 nM), which was 20-fold less potent than tacrine (IC50 = 31 nM). Promisingly though, most of these compounds were significantly less toxic than tacrine. In addition, 6d maintained a 65.5% viability in HepG2 cells at 1 mM, 6-fold less toxic than tacrine (10.9% viability at 1 mM). Moreover, 6d improved upon the known antioxidant capacity of KA (ORAC assay; 4.79 and 2.51 Trolox equivalents for 6d and KA, respectively), and it showed a capable neuroprotection against oligomycin/rotenone and Aβ1-40 in SH-SY5Ycells at 3 µM. Molecular modeling of hAChE with 6d R-and S-enantiomers indicated an association of both with the PAS via π-π stacking with Trp286 and Tyr341, acting as a barrier to the active site of the enzyme ( Figure 5).
Additional key H-bonds were noted, including the primary hydroxyl group for both enantiomers with Tyr72 and the tacrine-like amino group with Tyr124 (R-enantiomer) or Asp74 (S-enantiomer) [69].  [69]). Important interactions include π-π stacking with Trp286 and Tyr341 and H-bonds of the primary hydroxyl group with Tyr72 and the tacrine-like amino group with Tyr124.

Pyranonaphthalene, Pyranoquinoline, and Pyranonaphthoquinone Tacrines
Pentacylcic pyranotacrines bearing a fused naphthalene, quinoline, or naphthoquinone moiety (Scheme 3A) have shown diverse anti-AD activity, including AChE inhibition, BACE1 inhibition, ROS scavenging, and metal chelation [71][72][73][74]. The pyranoquinoline tacrines were designed to incorporate the hydroxyquinoline moiety of cliquinol, a known antioxidant and Cu-chelator [72,75], while the pyranonaphthoquinone tacrines were designed to incorporate the known 1,4-naphthoquinone BACE1i scaffold [73,76]. The compounds were prepared through closely related two-step sequences relying on 4H-pyran construction from malononitrile, aryl aldehydes, and either 2-naphthol, 1-naphthol, 8-hydroxyquinoline, or 2-hydroxy-1,4-naphthoquinone to give intermediates 12 [71], 13a,c,e-p [71,72], and 14a-t [73,74] (Scheme 3B). In all cases, a subsequent Friedländer reaction with the appropriate cycloalkanone and AlCl 3 gave the target compounds 9a-c, 10a-p, and 11a-t as racemic mixtures. with the appropriate cycloalkanone and AlCl3 gave the target compounds 9a-c, 10a-p, and 11a-t as racemic mixtures. In terms of biological activity, these compounds showed a weak inhibition of AChE with varying secondary activity. Among pyranonaphthalene and pyranoquinoline tacrines bearing a 1-methyl-1Himadazol-2-yl substituent 9a-c and 10a-d, compared to tacrine (EeAChE IC50 = 89.8 nM), all were weaker inhibitors of AChE. Even the most potent inhibitor 9b (EeAChE IC50 = 6.73 µM) was 75-fold less potent than tacrine. Non-competitive inhibition was indicated by the Lineweaver-Burk plot and confirmed by molecular modeling, which showed both enantiomers of 9b favoring an interaction with the PAS. The antioxidant capacity was promising, however, with compounds exhibiting between 1.47-2.75 Trolox equivalents by the ORAC assay, but 9b did show increased hepatotoxicity compared to tacrine in HepG2 cells [71]. Among 10e-p, 10l bearing a 3-methoxyphenyl substituent was the most potent against EeAChE (IC50 = 40 nM, compared to tacrine IC50 = 30 nM). SAR indicated the phenyl substituted inhibitors to be more potent than the pyridinyl substituted ones. The Lineweaver-Burk plot showed that 10l acted as a noncompetitive inhibitor of hAChE. Molecular modeling predicted that the R-enantiomer bound to hAChE at the PAS primarily through π-π stacking interactions. Moreover, noted were two H-bonds between the 8-amino group and Asp74 and Leu76. The S-enantiomer was predicted to bind in a similar fashion. These compounds showed In terms of biological activity, these compounds showed a weak inhibition of AChE with varying secondary activity. Among pyranonaphthalene and pyranoquinoline tacrines bearing a 1-methyl-1H-imadazol-2-yl substituent 9a-c and 10a-d, compared to tacrine (EeAChE IC 50 = 89.8 nM), all were weaker inhibitors of AChE. Even the most potent inhibitor 9b (EeAChE IC 50 = 6.73 µM) was 75-fold less potent than tacrine. Non-competitive inhibition was indicated by the Lineweaver-Burk plot and confirmed by molecular modeling, which showed both enantiomers of 9b favoring an interaction with the PAS. The antioxidant capacity was promising, however, with compounds exhibiting between 1.47-2.75 Trolox equivalents by the ORAC assay, but 9b did show increased hepatotoxicity compared to tacrine in HepG2 cells [71]. Among 10e-p, 10l bearing a 3-methoxyphenyl substituent was the most potent against EeAChE (IC 50 = 40 nM, compared to tacrine IC 50 = 30 nM). SAR indicated the phenyl substituted inhibitors to be more potent than the pyridinyl substituted ones. The Lineweaver-Burk plot showed that 10l acted as a noncompetitive inhibitor of hAChE. Molecular modeling predicted that the R-enantiomer bound to hAChE at the PAS primarily through π-π stacking interactions. Moreover, noted were two H-bonds between the 8-amino group and Asp74 and Leu76. The S-enantiomer was predicted to bind in a similar fashion. These compounds showed a moderate antioxidant ability, and 10l, although not the best antioxidant tested, gave an ORAC assay result of 1.83 Trolox equivalents.

Other Pyranotacrines
Other tri-and tetracylcic pyranotacrines with a diverse anti-AD activity have been studied (Scheme 4A) [71,[77][78][79]. The 4H-pyran core was formed by either a one-step reaction of ethyl benzoylacetate with benzaldehyde derivative and malononitrile in the presence of piperidine (in the case of 17a-p [77]) or a two-step sequence in which the appropriate imidazole-or quinolinecarboxaldehyde was first condensed with malononitrile and then treated with a 1,3-dicarbonyl (in the case of 17q,t,u,x-ab, and 18a,c) in the presence of piperidine (Scheme 4B,C) [71,78]. Alternatively, for the sulfamoyl-containing pyranotacrines 16f-t, SOCl 2 and microwave irradiation was first used to make p-sulfamoylbenzoyl chloride from the corresponding acid. Esterificaton with hydroxybenzaldehydes then gave sulfamoylbenzoate benzaldehydes, which were subjected to a three-component Aldol-Michael-cyclization sequence with malononitrile and dimedone under microwave irradiation to give intermediates 18f-j (Scheme 4D) [79]. In all cases, a final Friedländer reaction with the appropriate cycloalkanone in the presence of AlCl 3 gave the target compounds 15a-ad and 16a-t as racemic mixtures (Scheme 4B-D).
Eghtedari et al. found that all compounds 15a-p showed IC 50 values (EeAChE) < 6 µM. The best inhibitor 15i, bearing a R 1 3-bromophenyl substituent was 5-fold more potent than tacrine with an IC 50 = 69 nM and showed a mixed-type inhibition based on the Lineweaver-Burk plot. SAR suggested that the best inhibition was achieved with a R 1 2-or 3-bromo/chlorophenyl substituent. Molecular modeling predicted 15i to bind to the CAS of AChE near the catalytic triad. The predominant binding interaction was predicted to be the hydrophobic/π-π interactions, but an H-bond between the amino group and His440 was also noted. While 15i was predicted to cross the BBB (0.80 probability by the online admetSAR server), it was only weakly neuroprotective against H 2 O 2 -induced injury in PC12 cells and was moderately toxic to HepG2 cells (but still less toxic than tacrine) [77]. All synthesized sulfamoyl-containing pyranotacrines 16f-t were potent EeAChEi (IC 50 < 150 nM), but 16l was the most potent (IC 50 = 10 nM) and was 5.5-fold more potent than tacrine (IC 50 = 55 nM) ( Table 2). In general, the cyclohexyl moiety was favored over the cyclopentyl and cycloheptyl derivatives, and the p-sulfamoylbenzoate moiety was favored at the 4-position among this series. However, no molecular modeling or X-ray crystallography was performed to visualize the interaction of the inhibitors with the enzyme. Of the 15 compounds evaluated, 10 showed some degree of antioxidant activity [79]. Eghtedari et al. found that all compounds 15a-p showed IC50 values (EeAChE) < 6 µM. The best inhibitor 15i, bearing a R1 3-bromophenyl substituent was 5-fold more potent than tacrine with an IC50 = 69 nM and showed a mixed-type inhibition based on the Lineweaver-Burk plot. SAR suggested that the best inhibition was achieved with a R1 2-or 3-bromo/chlorophenyl substituent. Molecular modeling predicted 15i to bind to the CAS of AChE near the catalytic triad. The predominant binding However, modification to the imidazo-or quinolinopyranotacrines was found to weaken the inhibition. For example, compared to tacrine (EeAChE IC 50 = 89.8 nM), all imidazopyranotacrines 15q-t and 16a,b (Table 2) were weaker inhibitors of AChE [71]. For the quinolinopyranotacrines, constructed due to the prevalence of the 2-chloroquinolin-3-yl moiety in many pharmacologically active compounds, the most potent AChEi were 15x and 15ac (EeAChE IC 50 = 480 and 470 nM, respectively, compared to tacrine IC 50 = 190 nM) [78]. While lacking as AChEi, imidazo-and quinolinopyranotacrines have other beneficial properties. All of the imidazopyranotacrines showed high antioxidant capacity, with all but one compound exhibiting between 1.70-2.34 Trolox equivalents by the ORAC assay. Imidazopyranotacrine 15t, despite a weak AChE inhibition (IC 50 = 38.7 µM), was particularly promising given its high antioxidant capacity and low hepatotoxicity compared to tacrine in HepG2 cells (non-toxic at 1 mM) [71]. In addition, 15ac was able to significantly inhibit EeAChE-induced Aβ 1-40 aggregation, and both 15x and 15ac were less hepatotoxic than tacrine in HepG2 cells, with 15x being 27-fold less toxic. Moreover, 15x and 15ac showed promising neuroprotection in SH-SY5Y cells against oxidative stress, Aβ 1-40 aggregation, and tau-phosphorylation [78].
Modification to these N-heterocyclic tacrine scaffolds generally comes with a reduction in AChE inhibition and mixed other properties. For example, all pyridinotacrines 19a-j reported by Boulebd et al. [80] showed a weaker inhibition of EeAChE than tacrine (IC 50 = 30 nM), albeit over a fairly narrow range with IC 50 = 310-620 nM. While not the most potent AChEi, compound 19d (IC 50 = 500 nM) is noteworthy in that it showed no toxicity to HepG2 cells at concentrations as high at 1 mM. The authors hypothesized that reduced toxicity compared to tacrine was due to the fully substituted pyridine ring (no C-H bonds), which blocked oxidation to the reactive iminoquinone metabolite associated with hepatotoxicity in tacrine. However, none of the synthesized compounds displayed significant antioxidant activity [80]. Indolotacrine 20b showed that hAChE IC 50 = 1.5 µM, making it more potent than 7-methoxytacrine (IC 50 = 10 µM) but less potent than tacrine (IC 50 = 320 nM). In addition, 20b showed a promising inhibition of MAO-A (IC 50 = 490 nM) and was predicted to cross the BBB (PAMPA-BBB, P e = 6.6 × 10 −6 cm/s), but it lacked antioxidant activity and was more cytotoxic to a CHO-K1 cell line and more hepatotoxic to a HepG2 cell line than both tacrine and 7-methoxytacrine [81]. Lastly, compared to tacrine (hAChE IC 50 = 374 nM), quinoxalinotacrine 21 was a significantly weaker hAChE inhibitor (IC 50 = 22.0 µM). Molecular modeling predicted 21 interacted with hAChE in the mid-gorge region with the cyclohexyl ring oriented toward the CAS forming alkyl-π interactions with Trp86 and the phenyl and pyridinyl rings oriented towards the PAS forming π-π interactions with Tyr341 and Tyr337, respectively. Moreover, noted were key H-bonds between the amino group and pyrazine nitrogen and Tyr124 and Asp74. An initial in silico analysis predicted 21 had favorable ADME properties for CNS action (MW < 450, hydrogen-bond donors < 3, hydrogen-bond acceptors < 7, number of hydrogen bond donor < 5, Van der Waals surface area of polar nitrogen and oxygen atoms < 90, number of rotatable bonds < 8, hydrogen bonds < 8, logBB = −0.332) and that 21 (and 10 possible metabolites) showed no potential hepatotoxicity. Subsequent screening in HepG2 cells showed that 21 first showed a significant reduction in cell viability at 300 µM (for comparison, tacrine showed a significant reduction in cell viability at 30 µM). Additionally, 21 showed neuroprotection in SH-SY5Y cells against ROS (oligomycin A/rotenone-induced) and tau hyperphosphorylation (okadaic acid-induced) at concentrations as low as 0.1 µM, but the effect diminished above 1 µM [82]. cyclization with malononitrile, which, upon the tFriedländer reaction with cyclohexanone and AlCl3 gave the desired compounds (Scheme 5C) [81]. Quinoxalinotacrine 21 was prepared from the Friedländer reaction between 3-amino-2-quinoxalinecarbonitrile [83] and cyclohexanone in the presence of AlCl3 [82]. Modification to these N-heterocyclic tacrine scaffolds generally comes with a reduction in AChE inhibition and mixed other properties. For example, all pyridinotacrines 19a-j reported by Boulebd et al. [80] showed a weaker inhibition of EeAChE than tacrine (IC50 = 30 nM), albeit over a fairly narrow range with IC50 = 310-620 nM. While not the most potent AChEi, compound 19d (IC50 = 500 nM) is noteworthy in that it showed no toxicity to HepG2 cells at concentrations as high at 1 mM.
Compounds 22a-o were shown to be potent AChEi, as all compounds presented IC 50 values (enzyme source not specified) between 4.06-6.87 nM that are comparable to donepezil (IC 50 = 7.23 nM). There seems to be little difference between the pyrolo-, pyrazolo-, and furanotacrines in regards to the inhibitory activity, and no additional biological properties were examined [84]. A more in depth analysis was performed on pyrazolophthalazine tacrines 23a-u. Of the compounds synthesized, five showed very potent inhibition (EeAChE IC 50 < 100 nM) with 23o (IC 50 = 23 nM), bearing a m-fluorophenyl substituent, and 23l (IC 50 = 49 nM), bearing an o-methoxyphenyl substituent, being the most potent and being 16-fold and 7.5-fold more potent than tacrine, respectively (Table 3). SAR showed that the phenyl substituted compounds outperformed the alkyl substituted ones, and o/m/p-substituted phenyl substituents outperformed the non-substituted phenyl counterpart. The Lineweaver-Burk plot showed that 23l as a mixed-type inhibitor, and theoretical calculations predicted it to be CNS active (0.9260 probability by the online admetSAR server). Molecular modeling showed the R-enantiomer of 23l to interact more favorably with TcAChE, specifically at the PAS via the π-π stacking with Tyr120 and Trp278 and H-bonding with Tyr69. Additionally, 23l showed that the moderate ability to inhibit both self-and AChE-induced Aβ aggregation, was less toxic than tacrine with HepG2 cell viability remaining high (83%) at 300 µM, and exhibited a slight antioxidant activity as it protected PC12 cells from H 2 O 2 -induced death at 100 µM [85].  Compounds 22a-o were shown to be potent AChEi, as all compounds presented IC50 values (enzyme source not specified) between 4.06-6.87 nM that are comparable to donepezil (IC50 = 7.23 nM). There seems to be little difference between the pyrolo-, pyrazolo-, and furanotacrines in regards to the inhibitory activity, and no additional biological properties were examined [84]. A more in depth analysis was performed on pyrazolophthalazine tacrines 23a-u. Of the compounds synthesized, five showed very potent inhibition (EeAChE IC50 < 100 nM) with 23o (IC50 = 23 nM), bearing a m-fluorophenyl substituent, and 23l (IC50 = 49 nM), bearing an o-methoxyphenyl substituent, being the most potent and being 16-fold and 7.5-fold more potent than tacrine,  24a-d, 25a,b, 26a-e, and 27a,b have been very recently reported (Scheme 7A) [86]. The design rationale for these compounds was to merge the 4-chlorophenyltetrahydroquinoline moiety for AChE inhibition with the pyrazole and thiourea moieties of known COX-2 inhibitors (e.g., celecoxib) to potentially modulate the ACh and ROS/inflammatory aspects of AD. Starting from previously prepared amino pyrazolotacrine intermediate 28 [87], condensation with benzaldehyde derivatives or phenyl isothiocyanates gave the imines 24a-d and thioureas 25a,b. A further reaction of 25a,b with phenacyl bromides or ethyl bromoacetate afforded the thiazolidines 26a-e and thiazolidinones 27a,b, respectively (Scheme 7B) [86]. Interestingly, the authors chose to assess in vitro AChE inhibition through a percentage increase in contraction of frog's Rectus abdominis, as opposed to the more common Ellman assay [60], making a direct comparison to other compounds described in this review difficult. Suffice it to say, all compounds showed AChE inhibitory activity that was comparable or better than tacrine except 25a. In particular, 24b, 26e, and 27a,b were all roughly at least twice as active as tacrine. COX-2 inhibition assays indicated that 24b, 26e, and 27a,b showed IC 50 values between 0.76-0.89 µM, which was comparable to celecoxib (IC 50 = 0.84 µM). Of note, 26e showed remarkable selectivity towards COX-2 over COX-1 (13-fold more potent towards COX-2, better than celecoxib), which is beneficial for reducing adverse renal and gastrointestinal side effects [88]. The hepatoxicity was investigated through determination of serum glutamic pyruvic transaminase (SGPT) levels, and all compounds proved less hepatotoxic than tacrine. Molecular modeling with hAChE showed similar interactions for all compounds. For example, 27a,b (Figure 6) was predicted to interact in the PAS with the tricyclic core showing favorable hydrophobic interactions with Tyr341, Tyr337, Phe338, and Val294. The 4-chlorophenyl substituent also showed hydrophobic interactions with Tyr341 and Trp286, and the quinolinyl nitrogen was predicted to H-bond with Arg296. The thiazolidinone moiety showed a favorable interaction with Glu292 and Ser293 [86].  of pyrazolotacrines 24a-d, 25a,b, 26a-e, and 27a,b [86]. (B) Synthesis of  pyrazolotacrines 24a-d, 25a,b, 26a-e, and 27a,b. Reagents and conditions: (i) R-CHO, piperidine, EtOH, Δ, 4 h, 80-90%; (ii) R1-NCS, EtOH, Δ, 4-6 h, 83-89%; (iii) R2-COCH2Br, NaOAc, 1,4-dioxane, Δ, 4-6 h, 60-92%; (iv) ethyl bromoacetate, NaOAc, 1,4-dioxane, Δ, 4-6 h, 68-79%.

Urea and Thiourea Tacrines
Still further replacement of the aromatic A-ring of tacrine with additional heterocycles has resulted in thiourea and urea tacrines (Scheme 8A) [89,90]. Thiourea tacrines 28a-l were designed to combine tacrine and 3,4-dihydropyrimidin-2(1H)-thiones, known as calcium channel blockers [91,92] that have also shown metal-mediated, Aβ-related neuroprotection [93], into a multifunctional scaffold. The 3,4-dihydropyrimidin-2(1H)-thione moiety 34a-l was prepared by the reaction of substituted arylidenemalononitriles with thiourea in the presence of NaOMe. The exocyclic nitrile and amine then readily underwent the Friedländer reaction with cyclohexanone in the presence of AlCl 3 to afford the target compounds as racemic mixtures (Scheme 8B) [89]. The synthesis of 29a-c and 30-33 was done in one-step from the 2-amino-3-cyanotetrahydroquinoline 35. Precursor 35 could be condensed with aryl isothiocyanates to afford cyclized thiourea tacrines 29a-c, or it could be fused with urea or thiourea under high temperature (300 • C) to afford cyclized urea and thiourea tacrines 30 and 31 or moderate temperature (200 • C) to afford the open chain urea and thiourea tacrines 32 and 33 (Scheme 8C) [90].
Thiourea tacrines 28a-l showed a promising biological profile. The most potent hAChE inhibitor was 28k (IC 50 = 37.3 nM), which had a 3-bromophenyl substituent and was 10-fold more potent than tacrine (IC 50 = 374 nM). In addition, 28e, which had a 3-methoxyphenyl substituent, was the next most potent but significantly weaker (IC 50 = 3.05 µM). All other compounds in this series were significantly weaker AChEi (IC 50 > 5 µM). Both 28e and 28k showed non-competitive inhibition by Lineweaver-Burk plot analysis, suggesting PAS interaction. This was confirmed by molecular modeling with hAChE where (R)-28k was predicted to bind to the PAS by π-π interactions with the phenyl ring and Trp286 and Tyr72 and the aminopyridine ring and Tyr341. H-bonds were also seen between the tacrine-like amine and Asp74, and the bromo substituent was involved in halogen bonds. Overall binding of the S-enantiomer was similar. Notably, 28e was also predicted to bind to the PAS, but it lacked any binding contribution from the methoxy substituent, which may explain its reduced activity. In addition, compared to known calcium channel blocker nimodipine (49.62%), both 28e and 28k showed similar inhibition of Ca 2+ influx (30.40% and 42.23%), but neither 28e nor 28k showed significant inhibition of Aβ 1-42 self-induced aggregation. In addition, 28e was particularly promising in that it displayed no hepatotoxicity to HepG2 cells at concentrations up to 300 µM [89]. For 29a-c and 30-33, Ragab et al. chose to assess the in vitro AChE inhibition again through the less common percentage increase in contraction of frog's Rectus abdominis, making direct comparison to other compounds described in this review difficult. However, it is still noted that all compounds showed promising AChE inhibitory activity except compound 29b. Hepatoxicity was investigated through the determination of SGPT levels and glutathione (GSH) levels, and all compounds proved less hepatotoxic than tacrine. In silico calculations predicted that all compounds had promising drug-like characteristics [90]. resulted in thiourea and urea tacrines (Scheme 8A) [89,90]. Thiourea tacrines 28a-l were designed to combine tacrine and 3,4-dihydropyrimidin-2(1H)-thiones, known as calcium channel blockers [91,92] that have also shown metal-mediated, Aβ-related neuroprotection [93], into a multifunctional scaffold. The 3,4-dihydropyrimidin-2(1H)-thione moiety 34a-l was prepared by the reaction of substituted arylidenemalononitriles with thiourea in the presence of NaOMe. The exocyclic nitrile and amine then readily underwent the Friedländer reaction with cyclohexanone in the presence of AlCl3 to afford the target compounds as racemic mixtures (Scheme 8B) [89]. The synthesis of 29a-c and 30-33 was done in one-step from the 2-amino-3-cyanotetrahydroquinoline 35. Precursor 35 could be condensed with aryl isothiocyanates to afford cyclized thiourea tacrines 29a-c, or it could be fused with urea or thiourea under high temperature (300 °C) to afford cyclized urea and thiourea tacrines 30 and 31 or moderate temperature (200 °C) to afford the open chain urea and thiourea tacrines 32 and 33 (Scheme 8C) [90].

Amido-, Amino-, and Iminotacrines
Amidotacrines 36a-p were designed to explore the effect of adding an amide moiety to the 2-position of the cyclohexyl C-ring of tacrine, an underexplored SAR [94]. Meanwhile, amino-(37 and 39a-e) and iminotacrines 38a,b focus on replacing the aromatic A-ring of tacrine with amine or imine moieties (Scheme 9A) [90]. Synthetically, amidotacrines were accessed via the key intermediate ethyl 2-tacrine carboxylate (40a) or the corresponding 6-bromo analog 40b, which were prepared by the Friedländer reaction with 2-aminobenzonitriles, ethyl 4-oxocyclohexanecarboxylate, and BF 3 ·Et 2 O (Scheme 9B). To investigate the effect of different heterocyclic substituents at the 6-position, 40b was subjected to Pd-catalyzed Suzuki-Miyaura cross-coupling with the corresponding pyrazoloor pyrimido-boronic ester to give 40k and 40n, respectively. Nucleophilic acyl substitution of 40a/b with methylamine or hydrazine afforded 36a-d. Alternatively, 40a,b,k,n could be saponified to acids 41a,b,k,n and coupled to various phenyalkylamines using T 3 P to afford 36e-n or coupled to 5-amino-1-methyl-1H-pyrazole using HATU to afford 36o,p [94]. Compound 35, for the synthesis of amino-and iminotacrines, could be hydrolyzed with 70% sulfuric acid to afford 37, condensed with aryl aldehydes or benzoic acid hydrazide to afford Schiff bases 38a,b or benzoylhydrazide 39a, and acylated with benzoyl chloride or alkylated with different alkyl halides to afford derivatives 39b-e (Scheme 9C) [90]. With two exceptions, all amidotacrines 36a-p were more potent than tacrine (EeAChE IC 50 = 94.69 nM), and the best inhibitor was 36g (IC 50 = 5.17 nM), which had no substituent at the 6-position of the tacrine core and a phenylpropylamide at the 2-position (Table 4). SAR indicated that the phenylpropylamide at the 2-position gave the best inhibition of AChE, the bromo substituent at the 6-position seemed to have little effect, and a heterocycle (pyrazolo or pyrimido) at the 6-position decreased inhibition. Of note, 36c with no substituent at the 6-position and the hydrazide at the 2-position was a potent inhibitor (IC 50 = 12.97 nM). Molecular modeling with TcAChE showed that most compounds investigated positioned the tacrine moiety in the CAS, exhibiting π-π stacking with Trp84 and Tyr337 and H-bonding to His447, and oriented the 2-position towards the PAS allowing the amides to make key contacts with residues there. Of note, 36g was positioned to allow the amide nitrogen to H-bond with Tyr124, and 36c was positioned to allow the hydrazide to H-bond with Tyr337 ( Figure 7). Compound 36c was particularly promising as it uniquely, among the compounds tested, showed no cytotoxicity to HEK-293 cells or HepG2 cells at concentrations up to 300 µM [94]. Aminoand iminotacrines 37, 38a,b, 39a-e, also prepared by Ragab et al. and tested in an uncommon AChEi assay (see above), showed promising AChE inhibitory activity with the exception of compounds 39a,b. Compound 39c, with a 2-benzylamino substituent, had the highest activity (nearly 2-fold more active than tacrine). Molecular modeling of 39c with hAChE showed H-bonding between the benzylamino NH and Asp93 and between the nitrogen of the CN group and Tyr96, as well as hydrophobic interactions with various amino acids. This compound also was less hepatotoxic than tacrine and predicted to have promising drug-like characteristics [90]. With two exceptions, all amidotacrines 36a-p were more potent than tacrine (EeAChE IC50 = 94.69 nM), and the best inhibitor was 36g (IC50 = 5.17 nM), which had no substituent at the 6-position of the tacrine core and a phenylpropylamide at the 2-position (Table 4). SAR indicated that the phenylpropylamide at the 2-position gave the best inhibition of AChE, the bromo substituent at the 6-position seemed to have little effect, and a heterocycle (pyrazolo or pyrimido) at the 6-position decreased inhibition. Of note, 36c with no substituent at the 6-position and the hydrazide at the 2position was a potent inhibitor (IC50 = 12.97 nM). Molecular modeling with TcAChE showed that most compounds investigated positioned the tacrine moiety in the CAS, exhibiting π-π stacking with Trp84 and Tyr337 and H-bonding to His447, and oriented the 2-position towards the PAS allowing the amides to make key contacts with residues there. Of note, 36g was positioned to allow the amide nitrogen to H-bond with Tyr124, and 36c was positioned to allow the hydrazide to H-bond with Tyr337 ( Figure 7). Compound 36c was particularly promising as it uniquely, among the compounds tested, showed no cytotoxicity to HEK-293 cells or HepG2 cells at concentrations up to 300 µM [94]. Amino-and iminotacrines 37, 38a,b, 39a-e, also prepared by Ragab et al. and tested in an uncommon AChEi assay (see above), showed promising AChE inhibitory activity with the exception of compounds 39a,b. Compound 39c, with a 2-benzylamino substituent, had the highest activity (nearly 2-fold more active than tacrine). Molecular modeling of 39c with hAChE showed H-bonding between the benzylamino NH and Asp93 and between the nitrogen of the CN group and Tyr96, as well as hydrophobic interactions with various amino acids. This compound also was less hepatotoxic than tacrine and predicted to have promising drug-like characteristics [90].

Naphthalene and Naphthoquinone Pyranopyrimidinones/Pyrimidinimines
Pyranopyrimidinones and pyranopyrimidinimines 42a-o, 43a-o, and 44a-l (Scheme 10A) focus on two key modifications to the tacrine scaffold to modulate AChE inhibition and antioxidant activity: (1) Replacement of the aromatic A-ring with a fused pyranonapthalene or pyranonaphthoquinone moiety and (2) change of the central aromatic B-ring from an aminopyridine to a pyrimidinone or pyrimidinimine [95][96][97]. Several examples of the former modification have been presented herein, but the latter remains underexplored. The transition to a pyrimidinone or pyrimidinimine is based on the fact that quinazolinone and quinazolinimine derivatives inspired by naturally occurring alkaloids structurally resemble tacrine (e.g., deoxyvasicinone) and have shown promising activity as AChEi [98][99][100][101][102]. The naphthalene pyranopyrimidinones 42a-i and 43a-i were prepared using a three-component, one-pot reaction with ethyl cyanoacetate, benzaldehydes, and 2-or 1-naphthol in the presence of piperidine to give 45a,d,g and 46a,d,g, followed by condensation with lactams in the presence of POCl 3 (Scheme 10B) [95]. Similarly, the pyranopyrimidinimies 42j-o and 43j-o were prepared using the same three-component, one-pot reaction with the exception of malononitrile in place of ethyl cyanoacetate to give 48a,d and 49a,d. Subsequent condensation with lactams in the presence of POCl 3 gave the desired compounds (Scheme 10C) [96]. Moreover, in a similar fashion, naphthoquinone pyranopyrimidinones 44a-l were prepared using a three-component, one-pot reaction with ethyl cyanoacetate, benzaldehydes, and 2-hydroxynaphthalene-1,4-dione in the presence of potassium phthalimide-N-oxyl [103] to give 47a-f. Condensation with lactams in the presence of POCl 3 as before gave the desired compounds (Scheme 10B) [97].
Compound 42e displayed a mixed-type inhibition by the Lineweaver-Burk plot analysis. Additionally, it displayed potent antioxidant activity by the ORAC assay (4.7 Trolox equivalents) and showed no toxicity in HepG2 cells at concentrations up to 1 mM, but its BBB permeability is questionable (PAMPA-BBB, P e = 3.6 × 10 −6 cm/s) [95]. For the pyranopyrimidinimines, SAR clearly showed the 3-methoxyphenyl substituent to be more potent than the phenyl, and the 2-naphthol derived compounds to be more potent than those derived from 1-naphthol. Compound 42n displayed noncompetitive inhibition by the Lineweaver-Burk plot analysis, which agrees with molecular modeling predicting binding of the S-enantiomer (but not R) of 42n with the mid-gorge region of hAChE (Figure 8). Key interactions include a π-π stacking between Trp86 and the benzochromeno moiety and an H-bond between Asp74 and the imino moiety. Additionally, 42n and 42o displayed potent antioxidant activity by the ORAC assay (3.4 and 3.6 Trolox equivalents, respectively), but only 42o showed a significant Aβ 1-42 self-aggregation inhibition at 10 µM (40.3%) [96]. All naphthoquinone pyranopyrimidinones displayed some degree of antioxidant activity by the ORAC assay (44a = 2.78 Trolox equivalents), but most displayed significant toxicity to HepG2 cells. In addition, 44a was the exception as it maintained 84.5% HepG2 cell viability at 300 µM and was 2-fold less toxic than tacrine at the same concentration [97]. Among these three related series of compounds, a strong inhibition of AChE, such as tacrine, was generally maintained. For example, 42e, containing a piperidine-fused ring, 3-methoxyphenyl substituent, and derived from 2-naphthol was the most potent of the naphthalene pyranopyrimidinones regarding EeAChE inhibition (IC50 = 30.5 nM) and was comparable to tacrine   . Key interactions noted are the π-π stacking between Trp86 and the benzochromeno moiety and an Hbond between Asp74 and the imino moiety.

Other Tacrines
This final section focuses on tacrine-based molecules with little structural similarity to those of previous sections. Huprines combine the aminoquinoline moiety of tacrine and the carbocyclic bridged moiety of huperine A (Scheme 11A), a naturally occurring sesquiterpene alkaloid AChEi. Of particular interest is huprine Y that has shown dramatic improvement compared to parent structures regarding AChE inhibition, while also showing neuroprotection against 3-nitropropionic acidinduced neurodegeneration in mice [104,105]. In constructing 2-methoxyhuprine (50) (Scheme 11A), Mezeiova et al. sought to maintain the beneficial properties of huprine Y while adding the reduced toxicity of 7-methoxytacrine by combining the carbocyclic bridged moiety and methoxyaminoquinoline moiety [106]. Synthetically, 1,3-dibromoadamantane was first fragmented to the ketone by reaction with NaOH at high temperature. Treatment with 5% Pd/C and H2 in EtOH then led to isomerization of the carbon-carbon double bond. Finally, the Friedländer reaction with 2amino-5-methoxybenzonitrile and AlCl3 afforded racemic 50, which was converted to the hydrochloride salt for biochemical testing (Scheme 11B). Regarding hAChE inhibition, 2methoxyhuprine (50) showed a mixed-type inhibition, as determined by the Lineweaver-Burk analysis, with an IC50 = 2.63 µM and was more nearly 4-fold more potent than the parent 7- . Key interactions noted are the π-π stacking between Trp86 and the benzochromeno moiety and an H-bond between Asp74 and the imino moiety.

Other Tacrines
This final section focuses on tacrine-based molecules with little structural similarity to those of previous sections. Huprines combine the aminoquinoline moiety of tacrine and the carbocyclic bridged moiety of huperine A (Scheme 11A), a naturally occurring sesquiterpene alkaloid AChEi. Of particular interest is huprine Y that has shown dramatic improvement compared to parent structures regarding AChE inhibition, while also showing neuroprotection against 3-nitropropionic acid-induced neurodegeneration in mice [104,105]. In constructing 2-methoxyhuprine (50) (Scheme 11A), Mezeiova et al. sought to maintain the beneficial properties of huprine Y while adding the reduced toxicity of 7-methoxytacrine by combining the carbocyclic bridged moiety and methoxyaminoquinoline moiety [106]. Synthetically, 1,3-dibromoadamantane was first fragmented to the ketone by reaction with NaOH at high temperature. Treatment with 5% Pd/C and H 2 in EtOH then led to isomerization of the carbon-carbon double bond. Finally, the Friedländer reaction with 2-amino-5-methoxybenzonitrile a mixed-type inhibition for 51a, and both 51a,b showed decreased hepatotoxicity compared to tacrine [107].
Going forward, we believe that continued research in this field is vital in the fight against AD. The most promising compounds identified should be further tested in animal models to evaluate if promising in vitro properties are maintained. The pharmacokinetic properties of the most promising compounds should also be further investigated. For example, the PAMPA-BBB data presented in initial screening results may be altered in an in vivo system, which would necessitate additional chemical modification to enhance pharmacokinetics. There is an additional chemical space that remains to be explored, and we envision additional heterocyclic fusions to the tacrine core to further explore SAR.
As of yet, no "magic bullet" inhibitor has been identified. However, with each compound of this class that is synthesized and tested in combo, we gain incremental knowledge into the complex etiology and progression of AD that remains to be fully understood.